Vaccines are one of the most efficacious human health interventions available and protect against a very broad spectrum of infectious diseases. However, protective or therapeutic immunity still can not be raised against a number of latent and chronic pathogens. Conventional approaches that mainly elicit antibody responses have not been successful. Reasons include that epitopes may be variable, frequently masked or protected by microbial decoys, or because the pathogen hides in a way not accessible to antibodies.
Compared to vaccination with inactivated virions or purified subunits, live vaccines induce a broad response that also involves the cellular compartment of the immune system. However, due to the increased numbers of immunocompromized individuals and expansion of international mobility, the use of replication-competent strains can be associated with risks such as reversion to more pathogenic forms (Zurbriggen et al. 2008 in Appl Environ Microbiol 74, 5608-5614) or are severe adverse events both in recipients and contact persons of vaccinees (Kemper et al. 2002 in Eff Clin Pract 5, 84-90; Parrino and Graham 2006 in J Allergy Clin Immunol 118, 1320-1326).
Modern vectored vaccines (Excler et al. 2010 in Biologicals 38, 511-521; Plotkin 2009 in Clin Vaccine Immunol 16, 1709-1719) combine the advantages of an attenuated infection with the strong safety profile inherent to host-restricted vectors that can not replicate in the human or animal recipient. Especially promising hyperattenuated vectors are host-restricted poxviruses including the Modified Vaccinia Ankara (MVA) virus. The hyperattenuated poxviruses have demonstrated safety in clinical trials (Cebere et al. 2006 in Vaccine 24, 417-425; Dorrell et al. 2007 in Vaccine 25, 3277-3283; Gilbert et al. 2006 in Vaccine 24, 4554-4561; Mayr 2003 in Comp Immunol Microbiol Infect Dis 26, 423-430; Webster et al. 2005 in Proc Natl Acad Sci USA 102, 4836-4841) and yet are efficient stimulators of the immune response (Drillien et al. 2004 in J Gen Virol 85, 2167-2175; Liu et al. 2008 in BMC Immunol 9, 15; Ryan et al. 2007 in Vaccine 25, 3380-3390; Sutter and Moss 1992 in Proc Natl Acad Sci USA 89, 10847-10851; Sutter et al. 1994 in Vaccine 12, 1032-1040). Particularly, the MVA virus is related to Vaccinia virus, a member of the genera Orthopoxvirus in the family of Poxviridae. The MVA virus has been generated by 516 serial passages on chicken embryo fibroblasts of the Chorioallantois Vaccinia Ankara (CVA) virus. In the course of the attenuation process by repeated passaging to chicken derived material as production substrate, the MVA virus has lost approximately 15% of the genomic DNA at multiple sites (Mayr and Munz 1964 in Zentralbl Bakteriol Orig 195, 24-35; Meyer et al. 1991 in J Gen Virol 72 (Pt 5), 1031-1038). The MVA virus has been analysed to determine alterations in the genome relative to the wild-type CVA strain. Six major deletions of genomic DNA (deletion I, II, III, IV, V, and VI), totalling 31.000 base pairs, have been identified (Meyer, et al. 1991 in J Gen Virol 72 (Pt 5), 1031-1038). It became severely host cell restricted to avian cells. Whereas parental vaccinia virus has a broad host range, the MVA virus has a very narrow host range. For example, MVA does not replicate in human and non-human primate cells. In the human HeLa cell line, the replication block appears to occur at a defined step in genome packaging (Sancho et al. 2002 in J Virol 76, 8318-8334). In addition, the cells lines HEK 293 and Vero are not a preferred production system. It was further shown in a variety of animal models that the resulting MVA virus was significantly avirulent (Mayr and Danner 1978 in Dev Biol Stand 41, 225-234). Additionally, the MVA strain has been tested in clinical trials as vaccine to immunize against the human smallpox disease (Mayr 2003 in Comp Immunol Microbiol Infect Dis 26, 423-430). These studies involved over 120.000 humans, including high risk patients, and proved that, compared to CVA, MVA had diminished virulence or infectiousness while it maintained good immunogenicity.
However, the provision of adequate supply of the MVA virus is challenging. On the one hand, the MVA virus has to be given at high doses because it replicates at very low levels or not at all in the recipient. On the other hand, the MVA virus production systems which are presently available are time-consuming and expensive and can not satisfy the needs of the pharmaceutical industry.
As mentioned above, research on and production of MVA depends on avian cells. Currently, vaccine strains adapted to avian hosts are produced only in embryonated chicken eggs or on fibroblasts prepared from such eggs. This technology is associated with further disadvantages including continuous flow of primary animal-derived material into a demanding clinical production process and costs for maintenance of SPF (specific pathogen free) donor flocks. Because time from collection of the embryonated eggs to production of the vaccine is short, testing for extraneous agents is performed on the final bulk (Philipp and Kolla 2010 in Biologicals 38, 350-351). Occasionally, complete vaccine lots have to be discarded when contamination is confirmed by quality testing (Enserink 2004 in Science 306, 385).
Recently, to facilitate industrial application and vaccine programs in developing or newly industrialized countries, the inventors of the present invention designed and generated a host cell line fully permissive for vaccine strains depending on avian substrates (Jordan et al. 2009 in Vaccine 27, 748-756). They also developed a highly efficient and fully scalable chemically-defined production process for these viruses (Jordan et al. 2011 in Biologicals 39, 50-58).
Here, for the first time and with the above technology at hand, the inventors characterized stable isolates of subsequent generations of an already adapted and hyperattenuated MVA virus on a cell substrate fully permissive for the same hyperattenuated virus under highly artificial conditions imposed by virus production in a chemically defined suspension culture. This is an unusual experiment and the result is surprising. As described in the Principles of Virology (ISBN-10: 1555814433), the motivation of serial passaging is generally to adapt viruses to substrates with initially low permissivity: “Less virulent (attenuated) viruses can be selected by growth in cells other than those of the normal host, or by propagation at non-physiological temperatures. Mutants able to propagate better under these selective conditions arise during viral replication. When such mutants are isolated, purified, and subsequently tested for pathogenicity in appropriate models, some may be less pathogenic than their parent”.
The above characterization resulted in the identification of novel MVA viruses with point mutations in structural proteins. This result is consistent with virus propagation under artificial culturing conditions rather than selection within a certain host cell. The novel MVA viruses show beneficial properties in a chemically defined suspension culture compared to known MVA virus strains such as an increased infectious activity and a greater number of infectious units in the extracellular space. Said beneficial properties improve the industrial production of said MVA viruses. Particularly, they allow the production of the novel MVA virus strains in high yields. In addition, the novel MVA virus strains can be isolated directly from the cell-free supernatant which facilitates purification and, thus, the logistic and the operation of bioreactors producing said MVA viruses. This, in turn, reduces the costs of MVA virus production.